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Dexamethasone effects on induction of neoplastic transformation by Fujinami sarcoma virus in an in vitro chick embryo periosteal model for osteosarcoma.

Recently we have developed a model in vitro system for the study of factors regulating the histogenesis of osteosarcoma. In this system, Fujinami sarcoma virus (FSV) induces osteosarcomatous changes such as increased cell proliferation and altered patterns of bone and nonmineralized matrix (osteoid) formation. Such changes can be quantitated at the individual cell level, by computer-assisted morphometry. Here we report on the effects of dexamethasone (DEX) on FSV-induced neoplastic transformation and osteogenesis in chick embryonic periosteum cultures, as reflected by a series of histopathological parameters. Most significantly, it was found that compared to 10(-9) M DEX treated cultures, in 10(-7) M DEX pretreated cultures, the bone/osteoid ratio was increased due to a relative increase in the area of bone and a decrease in the area of osteoid. The number of [3H]thymidine-labeled cells decreased significantly, while the proportion of alkaline phosphatase positive cells increased. Double-label immunohistochemistry (with anti-P140gag-fps) and histochemistry for alkaline phosphatase activity was performed, to demonstrate production of the oncogene-encoded protein, and osteoblastic differentiation, respectively. In an in vitro transformation assay single cells derived from 10(-9) M DEX treated cultures formed a significantly higher number of colonies than those obtained from 10(-7) M DEX pretreated cultures. Taken together, the data indicate that in the chick embryonic periosteum culture system, pretreatment with 10(-7) M DEX inhibits the ability of FSV to induce neoplastic transformation. This effect is probably the result of DEX-induced cell differentiation, prior to infection with FSV.

Alkaline Phosphatase↗

Embryopathic effects of the oral hypoglycemic agent chlorpropamide in cultured mouse embryos.

OBJECTIVES: The oral hypoglycemic agent chlorpropamide was evaluated for embryopathic effects in the mouse embryo model. STUDY DESIGN: Mouse embryos (n = 10 to 31 per group) were exposed in whole embryo culture to serum from chlorpropamide-treated rats or chlorpropamide-supplemented control rat serum. Hypoglycemic serum from chlorpropamide-treated rats was supplemented with glucose to control for effects of hypoglycemia. Differences in malformation rates were evaluated by Fisher's exact test and numerical differences by analysis of variance. RESULTS: Mouse embryos were malformed by > or = 24 micrograms/ml of chlorpropamide, embryonic protein content and somites were decreased by > or = 130 micrograms/ml of chlorpropamide, and visceral yolk sac protein was reduced by > 500 micrograms/ml of chlorpropamide. Similar development occurred in serum from chlorpropamide-treated rats and chlorpropamide-supplemented control serum, and growth was not improved by adding glucose. CONCLUSIONS: Chlorpropamide produces malformations and growth retardation in mouse embryos in vitro at concentrations overlapping therapeutic levels in humans. Chlorpropamide-induced defects are not mediated by hypoglycemia or by products of chlorpropamide metabolism.

Animals↗

Elimination of the vitamin B12 uptake or synthesis pathway does not diminish the virulence of Escherichia coli K1 or Salmonella typhimurium in three model systems.

The role of iron in infection is of great importance and is well understood. During infection, both the host and the pathogen go through many complicated changes to regulate iron levels. Iron and vitamin B12 share certain features. For example, Escherichia coli has similar transport systems for both nutrients, and binding proteins for both are located in gastric juice, liver, saliva, granulocytes, and milk. It is because of such parallels between iron and B12 that we have explored the role of B12 in virulence. A btuB::Tn10 insertion which disrupts the gene encoding the vitamin B12 receptor from E. coli K-12 was P1 transduced into a virulent E. coli K1 strain. In both an infant-rat model and a chicken embryo model, no difference in virulence between the wild-type and the mutant strains was found. Strains of Salmonella typhimurium with mutations in the cobalamin synthesis pathway (Cob) and in btuB were used in a mouse model of virulence. Mutation of the Cob locus or of btuB does not decrease virulence. Interestingly, the inability to synthesize vitamin B12 actually increases virulence compared with the wild type in the S. typhimurium model. This effect is independent of the B12 intake of the mice.

Animals↗

Electrorotation of axolotl embryos.

The frequency dependent dielectric properties of individual axolotl embryos (Ambystoma mexicanum) were investigated experimentally utilizing the technique of electrorotation. Individual axolotl embryos, immersed in low conductivity media, were subjected to a known frequency and fixed amplitude rotating AC electric field and the ensuing rotational motion of the embryo was monitored using a conventional optical microscope. None of the embryos in the pregastrulation or neurulation stages of development exhibited any rotational motion over the field frequency range (10 Hz-5 MHz). Over the same frequency range, the embryos in the gastrulation stage of development exhibited both co-field and counterfield rotation over different ranges of the applied field frequency. Typically, the counterfield rotation exhibited a peak in the rotation spectrum at similar 1 KHz while the co-field peak was located at similar 1-2 MHz. The rotational spectral data was analyzed using a multishelled spherical embryo model to determine the electrical character of embryos during the early development stages (Stages 5-16; i.e., 16 cell through open neural plate stages).

Ambystoma mexicanum↗

Bacillus anthracis oedema toxin as a cause of tissue necrosis and cell type-specific cytotoxicity.

Oedema factor (OF) and protective antigen (PA) are secreted by Bacillus anthracis, and their binary combination yields oedema toxin (OT). Following PA-mediated delivery to the cytosol, OF functions as an adenylate cyclase generating high levels of cAMP. To assess OT as a possible cause of tissue damage and cell death, a novel approach was developed, which utilized a developing zebrafish embryo model to study toxin activity. Zebrafish embryos incubated with OT exhibited marked necrosis of the liver, cranium and gastrointestinal tract, as well as reduced swim bladder inflation. The OT-treated embryos survived after all stages of development but succumbed to the toxin within 7 days. Additional analysis of specific cell lines, including macrophage and non-macrophage, showed OT-induced cell death is cell type-specific. There was no discernible correlation between levels of OF-generated cAMP and cell death. Depending on the type of cell analysed, cell death could be detected in low levels of cAMP, and, conversely, cell survival was observed in one cell line in which high levels of cAMP were found following treatment with OT. Collectively, these data suggest OT is cytotoxic in a cell-dependent manner and may contribute to disease through direct cell killing leading to tissue necrosis.

Adenylyl Cyclases↗

Transperitoneal exchanges of water and solutes in the fetus with gastroschisis. Experimental study in the chick embryo.

Aiming at testing the hypothesis that, in the foetus with gastroschisis (GX) peritoneal dialysis during antenatal bowel exposure to the amniotic fluid may induce some modifications of the internal environment, we have studied the composition of blood, amniotic fluid and allantoic fluid in a chick embryo model of GX. Intestinal loops were surgically eviscerated in 184 embryos on the 14th day of incubation and exposed to the mixture of both embryonal fluids until the 19th day (hatching expected at the 21th). Typical lesions of GX were present in half of the 94 survivors (n = 42). For comparison we used a control group of non-manipulated animals (n = 19) and a "sham" group of animals in which the operation was carried out only until umbilical manipulation but without bowel exposure (n = 22). Chicks with GX were smaller and slightly dehydrated as attested by clinical signs and had moderately increased hematocrit. They had significant hyperkaliaemia and slight decreases in serum Na and Cl. Urea, total protein and protein fractions were not modified. Taking into account the gradients for such substances existing between the dialysate (amino-allantoic mixture) and the embryonal blood, the changes observed, albeit slight, suggest that there was some evidence of dialysis through the peritoneum: lower Na and Cl concentrations in the dialysate than in blood, induced slight falls in serum Na and Cl whereas higher K concentration resulted in severe hyperkaliemia. The high levels of albumin in the chick amniotic fluid did not allow protein depletion and therefore total protein and its fractions remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Abdominal Muscles↗

A quantitative approach to the study of cell shapes and interactions during early chordate embryogenesis.

BACKGROUND: The prospects of deciphering the genetic program underlying embryonic development were recently boosted by the generation of large sets of precisely organized quantitative molecular data. In contrast, although the precise arrangement, interactions, and shapes of cells are crucial for the fulfilment of this program, their description remains coarse and qualitative. To bridge this gap, we developed a generic software, 3D Virtual Embryo, to quantify the geometry and interactions of cells in interactive three-dimensional embryo models. We applied this approach to early ascidian embryos, chosen because of their simplicity and their phylogenetic proximity to vertebrates. RESULTS: We generated a collection of 19 interactive ascidian embryos between the 2- and 44-cell stages. We characterized the evolution with time, and in different cell lineages, of the volume of cells and of eight mathematical descriptors of their geometry, and we measured the surface of contact between neighboring blastomeres. These analyses first revealed that early embryonic blastomeres adopt a surprising variety of shapes, which appeared to be under strict and dynamic developmental control. Second, we found novel asymmetric cell divisions in the posterior vegetal lineages, which gave birth to sister cells with different fates. Third, during neural induction, differences in the area of contact between individual competent animal cells and inducing vegetal blastomeres appeared important to select the induced cells. CONCLUSIONS: In addition to novel insight into both cell-autonomous and inductive processes controlling early ascidian development, we establish a generic conceptual framework for the quantitative analysis of embryo geometry that can be applied to other model organisms.

Animals↗

Conception and realization of artificial dyed embryos for training in in vitro fertilization and embryo transfer (IVF and ET).

An artificial embryo model is proposed for current training in in vitro fertilization and embryo transfer. It consists in resin microballs of 180 micron diameter, stained either with fluoresceinamin or methylene blue. These microspheres release, even at the maximum dye concentrations tested, very negligible amounts of dye; heat stable they can be sterilized. This model, already checked for human embryo transfer, seems to be a useful tool in other reproduction studies such as embryo transport.

Education, Medical, Continuing↗

Decoding bipotency: a transient regulatory state bridging totipotency and lineage commitment.

Early mammalian embryogenesis entails a coordinated transition from totipotency to the first lineage bifurcation, giving rise to embryonic lineages and the extra-embryonic trophectoderm. The mechanisms by which totipotency is resolved into lineage-primed states remain incompletely understood. Emerging evidence supports a non-binary model in which cells traverse a continuum of potency states, passing through a transient bipotent intermediate that retains both embryonic and extra-embryonic potential while exiting totipotency. Here, we synthesize recent advances in the mechanisms that establish, maintain, and resolve bipotency. We emphasize the coordinated roles of transposable elements, transcription factors, and signaling pathways in regulating this transition. We also highlight newly developed bipotent stem cell models and their implications in generating advanced embryo models in vitro. Notably, current insights are largely derived from mouse systems; given key differences between mouse and human early embryogenesis, extending these findings to human models remains a critical next step.

Animals↗

Surgical approach of an early mammalian embryo: the rabbit model.

We describe an experimental surgical model in early rabbit embryo (12.5 days of gestation). Twenty-one embryos were operated, of which 9 survived the first postoperative week and 5 had normal further growth until term. As far as we can ascertain, this is the first successful report of a surgical approach in early mammalian embryos. We think that this model may be useful for workers involved in the study of early congenital malformations.

Animals↗

Danazol, a new steroidal inducer of delta-aminolevulinic acid synthetase.

Danazol (a derivative of 17 alpha-ethinyl testosterone) has recently been reported to cause clinical exacerbations of acute intermittent porphyria. In the present study, we demonstrate that danazol is an inducer of hepatic delta-aminolevulinic acid synthetase and that it increases hepatocyte porphyrin production using a chick embryo model. The findings provide an explanation for the clinical observations. Although danazol is a porphyrinogenic agent in the chick embryo and humans it has been reported to be ineffective as a porphyrinogen in rats. The present findings, therefore, also confirm the superiority of the chick embryo as a model system for the screening of drugs and chemicals which may have the potential to cause clinical exacerbations of porphyria.

5-Aminolevulinate Synthetase↗

Mathematical models of the embryo and fetus for use in radiological protection.

This development of new mathematical models arose from our current work in external neutron dosimetry for the embryo and fetus when pregnant women travel at commercial aircraft altitudes. A problem of concern in radiation protection is exposure of pregnant women to ionizing radiation because of the high radiosensitivity of the embryo and fetus. Special regulations and dosimetric considerations are necessary for pregnant women at the work place and in the public. To perform dosimetry, mathematical models for the embryo and the fetus, together with the modified adult female model for pregnant woman, are required. There are no models available for embryo. Models developed for the fetus need to be updated with the new reference values such as those in ICRP Publication 89. This article presents mathematical models for the embryo and fetus at different stages: the embryo at 8 wk and the fetus at the end of each trimester. In addition to fetal skeleton, the fetal brain is explicitly modeled because of its high radiosensitivity. All model parameters are determined from the most recent reference values available. The models are designed so that an interpolation can be easily performed to generate a model of embryo/fetus at any given stage of development. This feature also allows convenient adaptation of the models to different reference values representing various ethnic populations. The new mathematical models presented here were developed for external dosimetry. They can also be used for internal dosimetry purposes, if other organs inside the female phantom are adjusted accordingly.

Adult↗

Intra- and interstrain differences of virulence among nontypeable Haemophilus influenzae strains.

Nontypeable Haemophilus influenzae (NTHi) is sometimes the causative agent of invasive diseases, and it has been suggested that there may be differences in virulence among NTHi strains. Whilst studying clinical isolates of NTHi in a rat model of acute otitis media, intra- and interstrain differences in virulence were observed. Two strains with suddenly reduced capacity to cause middle ear infections and one highly virulent strain with dose requirements comparable only to encapsulated H. influenzae strains were further investigated, together with 15 other H. influenzae strains. The strains were characterized by analyzing the lipopolysaccharide, the outer membrane proteins, the hemagglutinating ability, and the polymerase chain reaction products after amplification of a gene sequence associated with encapsulation. The pathogenic capacity was assessed in two different in vivo models. It was found that the two strains with reduced pathogenic capacity could regain their virulence after animal passage. The LPS analysis and the results from the chicken embryo model suggested that the observed change in virulence might be associated with the lipopolysaccharide. For the non-animal-passaged strain 3655 there were indications that an undefined factor(s) contributed to its relatively potent virulence. As all three strains lacked genes necessary for encapsulation, in no case could any part of the increased virulence be attributed to the expression of small amounts of capsule.

Animals↗

The relationship between neural alterations and behavioral deficits after prenatal exposure to heroin.

The present studies employ multitudinous approaches in order to overcome the methodological obstacles in the understanding of the relationship between neurochemical alterations and behavioral deficits induced by heroin during prenatal development. Mice were exposed prenatally to heroin via daily subcutaneous injections of 10 mg/kg, on gestation days 9-18. At age 50 days, the heroin-exposed offspring displayed behavioral deficits as assessed in the eight-arm and Morris mazes, pointing to possible alteration in the septohippocampal cholinergic innervations. Biochemically there was increased presynaptic activity of these innervations as attested to by the increased [3H]hemicholinium-3 (HC-3) binding sites and by K+-stimulated inositol phosphate (IP) formation. Postsynaptically, there was global hyperactivation along the different components of the nerve conduction cascade, including an increase in M1 muscarinic receptor Bmax, a general increase in G-proteins (GP) including the most relevant, G subtype, and an increase in IP formation and in basal protein kinase C (PKC) activity. However, there was desensitization of PKC activity in response to cholinergic agonist in the heroin-exposed offspring. Transplantation of normal embryonic cholinergic cells to the impaired hippocampus reversed the behavioral deficits and both the pre- and postsynaptic hyperactivity and resensitized PKC activity. To support and further strengthen the findings of the neural grafting study, correlation of the heroin-induced behavioral deficits with the biochemical alterations, done within individuals, was applied. The results showed high r values for IP formation, basal PKC, and PKC desensitization. The r values for HC-3 binding were statistically significant but relatively low. Taken together, the findings of the neural grafting and correlation studies bring us closer to understanding the relationship between the prenatal heroin-induced biochemical and behavioral changes. However, mammalian models possess the inherent methodological hindrances, stemming from possible maternal effects. To provide a control for these confounding variables, a chick embryo model was applied in which filial imprinting, a behavior related to a specific hyperstriatal nucleus, served as an endpoint. Heroin was administered to developing chick embryos by injecting the eggs (20 mg/kg) on incubation days (ID) 0 or 5. Prehatch exposure to heroin markedly diminished the ability for filial imprinting in the hatched chicks.

Acetylcholine↗

Developmental bioinformatics: linking genetic data to virtual embryos.

This paper discusses current efforts to produce databases of gene expression for the major model embryos used in developmental biology. The efforts to build these resources were motivated by the need for immediate internet access to all types of research data, and the production of these databases is a major and new challenge for bioinformatics. Thus far bioinformatics has mainly been concerned with textually oriented resources and data, much of it concerned with gene and protein sequences. Because the genetic basis of developmental biology is integrated with developmental anatomy, these databases require the use of images to link molecular data with spatial information. In order to standardise database formats, digital atlases of some model systems are being produced that include integrated anatomical descriptions and these are being linked to appropriate genetic data. Integrating such image-based, searchable data into databases makes new demands on the field of bioinformatics and we consider here the imaging modalities that are used to obtain information and we discuss in particular the production of 3D images from serial sections. Next, we consider how to integrate textual and spatial descriptions of gene expression and the key tool needed to make this possible, i.e. anatomical nomenclature. A short review of internet resources on developmental biology is also given and future prospects for the development of these databases are discussed.

Animals↗